Center for Advanced Biotechnology and Medicine and the Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey 08854 United States.
Migal-Galilee Research Institute, Kiryat Shmona, 11016, Israel.
ACS Synth Biol. 2020 Dec 18;9(12):3400-3407. doi: 10.1021/acssynbio.0c00514. Epub 2020 Nov 13.
expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron-sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters . While reconstitution produces a mixture of oligomers that depends on solution conditions, production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S] cluster. The multinuclear cluster of assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron-sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.
金属蛋白的表达需要细胞内特定的金属运输和掺入机制。设计合成的金属蛋白通常在没有目标金属的情况下进行纯化，然后通过再构成引入目标金属。这个额外的步骤增加了通过高通量文库筛选或实验室进化进行蛋白质优化的复杂性。我们证明了一种设计的卷曲螺旋铁硫蛋白（CCIS）可以与[4Fe-4S]簇组装。虽然再构成会产生依赖于溶液条件的寡聚物混合物，但 生产会产生稳定的同源三聚体，配位一个单一的、抗磁性的[4Fe-4S]簇。组装的 CCIS 的多核簇对分子氧的降解更具抗性。只需要两个金属配位半位点中的一个，表明在招募金属簇时分子识别的特异性。CCIS 没有经过进化的影响，是研究铁硫蛋白生物发生和开发合成多核氧化还原酶的独特平台。
